ABSTRACT The goal of this research program is to develop an advanced, noninvasive, molecularly specific imaging tech- nology ? ultrasound-guided modulated photoacoustic imaging augmented by optically-activatable, targeted con- trast agents, capable of immediate, accurate, background-free assessment of pathologies in vivo. The underlying hypothesis of this project is that ultrasound-guided photoacoustic imaging of optically modulated contrast agents can be performed in real time, yielding immediate diagnostic information. The approach is based on the unique combination of an optically-activatable molecularly-targeted imaging contrast agent and corresponding laser/ul- trasound imaging device. Specifically, development of a highly-sensitive imaging contrast agent consisting of silica nanoparticles doped with optically-activatable photoabsorbers and coated by an antibody-functionalized lipid shell will be undertaken. The photoabsorbers embedded in the silica core exhibit ~50?s-lived, transient near-infrared optical absorption upon prior red laser irradiation. The contrast agent will be imaged using a clinical ultrasound (US) imaging system, interfaced with two (pump/probe) pulsed laser sources, operating in either ultrasound or photoacoustic imaging modes. Pump laser pulses will repeatedly activate the contrast agent, thus allowing for probe laser pulses to generate photoacoustic signal that can be processed to generate background- free modulated photoacoustic (mPA) images spatially co-registered with grayscale ultrasound (US) images. Fur- thermore, by probing different dark state lifetimes of optically activated contrast agents, multiple mPA ?colors? will be simultaneously imaged within tissue. These US-mPA images will display molecular and functional signa- tures of the disease within the structural content of the tissue. The specific objective of this project is to develop an ultrasound-guided modulated photoacoustic imaging approach and demonstrate the developed approach in the background-free detection of micrometastases in sentinel lymph nodes using a murine model of metastatic breast cancer. Indeed, one of the critical components of the clinical cancer management is the analysis of re- gional lymph nodes where current diagnostic methods including imaging suffer from low sensitivity and specificity. Therefore, this US-mPA system will be specifically designed for imaging micrometastases in sentinel lymph nodes. Improved sensitivity and specificity of US-mPA imaging will be demonstrated through synthesis of mo- lecularly targeted contrast agent and coordinated instrumentation development to maximize impact of these new materials and the imaging approach. The successful outcome of this study will enable design and development of a clinical imaging system and contrast agents for background-free molecular imaging of various pathologies.